Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > DNA Techniques > DNA Extraction Forum
Register Search Today's Posts Mark Forums Read

DNA Extraction Forum DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


DNAse in DNA Isolation Interfering with In Vitro Transcription Reaction

DNAse in DNA Isolation Interfering with In Vitro Transcription Reaction - DNA Extraction Forum

DNAse in DNA Isolation Interfering with In Vitro Transcription Reaction - DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 06-23-2008, 12:51 AM
Pipette Filler
Points: 2,483, Level: 32 Points: 2,483, Level: 32 Points: 2,483, Level: 32
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2006
Posts: 17
Thanks: 0
Thanked 1 Time in 1 Post
Question DNAse in DNA Isolation Interfering with In Vitro Transcription Reaction



Hi, what i am doing is trying to remove all DNA template from my in vitro transcription reaction using T7 polymerase.

The DNAse is RNase free and DNAse digestion was at 37C but the problem is that in the first 20 minutes the DNA band intensity goes down when follow the DNA intensity on an agarose gel by time course!


Weirdly the DNA band intensity starts building up between 20minutes to 60minutes...

What is going on?

thanx
Reply With Quote
  #2  
Old 06-23-2008, 12:54 AM
Pipette Filler
Points: 2,385, Level: 31 Points: 2,385, Level: 31 Points: 2,385, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2006
Posts: 21
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: DNAse in DNA Isolation Interfering with In Vitro Transcription Reaction

Hi,
have you repeated this error? Can you show us an image of the gel?

The DNAse may be binding to the DNA and then interfering with gel migration and giving the appearance of reduced band intensity.


Possibly the DNAse was added at too high a concentration or the buffer in it affected the in vitro transcription.

I would try to order new DNase that is fresh DNAse from another company and try the experiment again.

Make sure you inactivate the DNAse first also.
Reply With Quote
Reply

Tags
dna , dnase , interfering , isolation , reaction , transcription , vitro


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Non-isotopic method for In vitro transcription cv2135@columbia.edu Protocols and Methods Forum 0 06-16-2008 02:15 PM
In vitro transcription problems AudioHELP Protocols and Methods Forum 2 12-17-2003 06:26 PM
T7 in vitro transcription Vayuputra Protocols and Methods Forum 0 07-19-2003 06:01 PM


All times are GMT. The time now is 12:24 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.11574 seconds with 16 queries